Enzymatic Breakage and Joining of Deoxyribonucleic Acid

Abstract Polynucleotide ligase of bacteriophage T4, in contrast to the DNA ligase of Escherichia coli, catalyzes the joining of oligomers of deoxyribonucleotides hydrogen-bonded to complementary strands of either polydeoxyribonucleotides or polyribonucleotides. Hybrid molecules in which oligomers of ribonucleotides are hydrogen-bonded to complementary strands of polydeoxyribonucleotides can also serve as substrates for the T4 enzyme. The rate of joining of oligomers in hybrid molecules of this type is only 1 to 2% of that observed when both strands are polydeoxyribonucleotides. No activity has been detected when both strands are polyribonucleotides. The products of these reactions have been characterized by sedimentation and end group analysis; in all cases joining occurs through phosphodiester bond formation. The activities toward hybrid molecules and toward DNA containing single strand breaks appear to be identical as indicated by: (a) identical ratio of the activities during a 1000-fold purification, (b) similar reaction requirements, and (c) identical thermosensitivity of both activities purified from cells infected with a mutant bacteriophage containing a temperature-sensitive ligase. It had been shown previously that the introduction of an rII mutation into phage T4 could suppress an amber mutation in the ligase gene. In these studies it is shown that the double mutant, T4rIIamH39X, carrying an amber mutation in the ligase gene, does not have detectable levels of T4 ligase in the nonpermissive host. Similarly, T4rIItsA80, carrying a temperature-sensitive mutation in the ligase gene, can grow at the nonpermissive temperature, despite retaining its temperature-sensitive ligase.